Team:XJTU-CHINA/Demonstrate
Demonstration
Our team aims to realize selective production of limonene and linalool. We reconstructed a red light-operating system used in 2018 team UCAS-China and built the controlling circuit to control the production of the two fragrances, while precursor circuit provides with a general precursor - GPP.
Our project was demonstrated to be functional in three parts.
- Precursor Circuit was proved to yield high level expression ofbe geranyl pyrophosphate synthase GPPs which will eventually lead to the efficient production of GPP.
- Two fragrance molecules can be successfully biosynthesized by the engineered E. coli strains with selected linalool synthase and limonene synthase in our project.
- Our designed light-controlled bidirectional switch is confirmed to be functional by quantitively and quantitatively measurement of GFP and RFP fluorescent intensity.
Validation of Precursor Circuit
In our study, we aim to achieve biosynthesis of limonene and linalool in E. coli DH5 . According to 2018 team GreatBay_China's results, no target product was detected using gas chromatography when shake-flask fermentation was conducted by this strain induced by 25 µM IPTG for 24 hours due to the lack of endogenous MVA pathways in E. coli. Thus we decided to co-express an MVA pathway together with GGP synthesis. 2018 team GreatBay_China generously gave us one plasmid pJBEI6409(Keasling et al, 2013), which contains a MVA pathway in addition to an GPPs-LS operon. We reconstructed this plasmid and get a plasmid only contains a series of enzymes from MVA pathway.
Precursor Circuit converts carbon sources like glucose to geranyl pyrophosphate(GPP), ready to be catalyzed into limonene and linalool. We constructed the circuit by modification of pJBEI6409. The 11523 bp-length vector is constructed based on pSB1C3 backbone and each operon is under the regulation of Ptrc, and the digestion verification result of two endonucleases was as follows.
Since the product of this circuit, GPP is unstable to be detected via HPLC etc., we performed SDS-PAGE analysis to compare the expression level of key enzyme--GGPs with and without IPTG induction to test if this circuit works properly, and the result is shown below. Click to see our protocol.
The results of SDS-PAG clearly demonstrate the high expression of the GPPs in the precursor circuit, indicating its functionality and eventual GPP synthesis.
Limonene and Linalool can be successfully synthetized
In production circuits, limonene synthase CS and three linalool synthases (LS)-LGF, DHJ, RXY under the control of Ptrc are ligated to pSB1K3 to validate the activity and function of these enzymes. And the pGPP plasmid and CS-harboring or LS-harboring was co-transformed into E. coli DH5α for limonene or linalool synthesis.
Ptrc and LS or CS genes was obtained by PCR flanked two Type IIS sites BsaI and was used for Golden Gate Assembly to construct the expression vectors. The length of limonene synthase and DHJ is 1632bp and 1695 bp respectively. Here are the gel results confirming the success amplifications.
The expression of limonene synthase and linalool synthases are under the regulation of Ptrc. RT-qPCR and SDS-PAGE were conducted to test their expressions, as well as Gas Chromatography analysis was used to detect limonene or linalool yields. Click to see our protocol.
We measured the transcription level of CS and LSs of four different strains, E. coli harboring only pGPP vector, E. coli with dual plasmid pGPP+pDHJ (Stain B in Fig 3), pGPP+pLGF (Stain C in Fig 3), pGPP+pCS (sStain A in Fig 3),and relative mRNA levelst compared with the blank strain only harboring pGPP are shown in Fig. 5.
As shown in Fig. 5, compared to blank strain, the relative mRNA level of three strains were much higher, which DHJ were most actively transcripted, and CS was relatively less, but all indicated the efficient transcription of these three genes in engineered E. coli.
We also conducted SDS-PAGE of whole cell proteins to ensure their expression. As shown in Fig 5, compared to blank strain only with pGPP vector, DHJ, LGF and CS protein was expressed but yields were not high, and because of the leaky expression, expression level with IPTG induction were just a little bit higher than the level without IPTG induction.
Both the results of above RT-qPCR and SDS-PAGE demonstrate the expression of the linalool synthase DHJ, LGF and limonene synthase CS.
GC analysis will confirm whether the expressed protein is functional. Firstly we analyzed a positive limonene-containing control that yielded a strong peak as indicated by the black line with the retention time abound 1.44 minutes. The results show that there is no overlap of retention time between n-Hexadecane and limonene, and indicates the GC method is reliable for testing.
In comparison, we expressed the limonene generator with trc promoter in DH5α E. coli and lysed the cell culture, extracted with n- hexadecane, and the extraction was subjected to gas chromatography mass spectrometry.
The results show that the retention time of limonene is much more clear at 1.37 minutes after optimization when the amount of limonene is small. It shares the same retention time our experimental result of samples from E. coli that is transformed with pGPP and pCS (Fig 6). And the important GC-MS result showed the MS profile is exactly the same with literature reported, further confidently confirming its production. This result indicates that GPP production can be achieved by precursor circuit, and most importantly, our limonene synthase can work properly to convert GPP to limonene even though the yield of limonene remains low, which needs to be further improved by more metabolic engineering of the strain.
The results indicate that the the MS profile is exactly the same with literature reported, confirming its production.
It is the same with linalool production. As shown in Fig 8, a small peak can be detected by GC, which also confirmed the function of our selected LS gene LGF, and further optimization is still needed.
All in all, the compressive analysis of RT-qPCR, SDS-PAGE and GC have demonstrated that two fragrance molecules limonene and linalool can be successfully synthetized by engineered E. coli with our selected enzymes.
Light-controlled bidirectional switch is functional
The light-fluorescent circuits (see circuits) were constructed by two-step Golden Gate Assembly and were confirmed by colony PCR. The length of this fragment is 1467 bp and the following results showed the successful constructions.
In order to validate the effectiveness of this switch, we qualitatively measure the fluorescent of this circuit at different time. Sample of DH5α E. coli transformed with BBa_K3052030 is incubated for 24 hours without red light and then continue incubating 24 hours with red light. We photographed both the sample of control group (DH5α E. coli wildtype) and experimental group(E. coli with light-fluorescent circuit ) by a fluorescence microscope at 24 hours (24 hours without light treatment) and 48 hours (24 h without light treatment and 24 h with red light treatment). The figure indicated that the bidirectional switch was functional to switch from GFP to RFP.
Furthermore, we used COFOCAL to sensitively detect GFP and RFP while switch is shifting. After the sample above had been incubated for 24 hours without light treatment, it was treated with intense red light (wavelength=640nm). After incubating for another 3 hours with red light treatment, we took out some of the sample and perform CONFOCAL imaging. The result shows that the RFP was appearing with red light treatment, which indicates this switch can be effectively triggered by red light.
To quantitatively test the efficiency of this light switch, we decided to cultivate the cells in dark environment first and then trigger the switch with intense red light. When they had been incubated for 30 hours without red light, we start to put intense red light around the medium. During the experiment, we measured the GFP and RFP intensity of the sample every 2 hours.
From the above figure we can see that the GFP intensity increased gradually while RFP intensity maintained to be at a low basic level without red light. But after red light treatment, GFP intensity dropped sharply to a low and stable value in 4 hours, and slow increase of RFP intensity was obviously observed. The results clearly indicates the effectiveness of this bidirection switch and the fast response time to the red-light stimulation.
All in all, the results quantitively and quantitatively demonstrated the light controlled switch is functional. Compared to the previous part which achieved the monodirectional regulations (BBa_K2598038 of UCAS_China 2018), our improved version can efficiently achieve bidirectional regulation of two genes, leading to more flexible and selective regulations. BBa_K3052030 in our project is the proof of concept of this bidirectional switch, and the fluorescent proteins will be alternated with our selected linalool synthase and limonene synthase which have also been proved to be functional, to achieve the selective production of linalool and limonene.